靶向干扰DNMT1基因慢病毒载体构建及其对结肠癌SW620细胞株生物学行为的影响  

Construction of lentivirus- mediated shRNA targeting DNMT1 and investigation of its effect on biological behavior in colon cancer SW620 cells

作　　者：董峰[1] 马君俊[1] 何子锐[1] 张鲁阳[1] 洪希周 

机构地区：[1]上海交通大学医学院附属瑞金医院普外科上海市微创外科临床医学中心,上海200025

出　　处：《临床和实验医学杂志》2016年第23期2303-2307,共5页Journal of Clinical and Experimental Medicine

摘　　要：目的构建干扰DNMT1基因的慢病毒并感染结肠癌SW620细胞株,观察其对细胞生物学行为的影响。方法合成DNMT1 shRNA寡核苷酸链,与p CDH-CMV-MCS-EF1-cop GFP连接构建重组慢病毒质粒,转染HEK293T细胞进行病毒包装。包装后的病毒感染SW620细胞,分为3组:Lentivirus-DNMT1组(LV-DNMT1)、Lentivirus-vector组(LV-vector)、空白对照组(Blank)。采用定量逆转录聚合酶链反应(RT-PCR)和蛋白质印迹法(Western Blot)检测细胞DNMT1及T-cadherin表达,MTT法和流式细胞术检测细胞增殖和凋亡,Transwell和细胞划痕实验检测细胞侵袭和迁移。结果成功构建了靶向干扰DNMT1的慢病毒载体。与LV-vector组和Blank组比较,LV-DNMT1组DNMT1 mRNA(0.49±0.09)和蛋白(0.39±0.11)表达显著下调(P<0.05),而T-cadherin mRNA(0.74±0.12)和蛋白(0.69±0.13)表达显著上调(P<0.05)。与LV-vector组和Blank组比较,LV-DNMT1组第12 h、24 h、36 h、48 h、72 h时的OD(570)值降低(P<0.05),细胞总凋亡率(26.06%)增加(P<0.05),穿膜细胞数目[(21.33±8.02)个]和细胞迁移距离[(303.27±47.58)μm]减少(P<0.05)。结论 DNMT1慢病毒载体可有效敲减DNMT1表达和上调T-cadherin表达,抑制SW620细胞增殖,促进其凋亡,降低其侵袭、迁移能力。Objective To construct lentivirus- mediated shRNA targeting DNMT1 and observe the effect on biological behavior in colon cancer SW620 cells. Methods DNMT1 shRNA oligonucleotide chain was synthesized and inserted into the lentivirus plasmid p CH- CMV- MCS- EF1- cop GFP. The DNMT1 shRNA recombinant plasmid was transfected into SW620 cells by Lipofectamine 2000. SW620 cells were infected by lentivirus after packaging,the experiment was divided into 5 groups： Lentivirus- DNMT1 group（ LV- DNMT1）,Lentivirus- vector group（ LV- vector）,Blank group（ Blank）. The expression of DNMT1 and T- cadherin were detected by RT- PCR and Western Blot,the proliferation and apoptosis of cells were detected by MTT assay and flow cytometry,the invasion and migration of the cells were detected by Transwell and cell scratch test. Results The lentivirus- mediated shRNA targeting DNMT1 was successfully constructed. Compared with LV- vector and Blank group,the DNMT1 mRNA（ 0. 49 ± 0. 09） and protein（ 0. 39 ± 0. 11） in LV- DNMT1 group were significantly down- regulated（ P〈0. 05）,the T- cadherin mRNA（ 0. 74 ± 0. 12） and protein（ 0. 69 ± 0. 13） in LV- DNMT1 group were significantly up- regulated（ P〈0. 05）. The OD（ 570 nm） values at 12 h,24 h,36 h,48 h,72 h in LV- DNMT1 group were decreased（ P〈0. 05）. The total cell apoptosis rate（ 26.06%） was increased,the number of crossed cells（ 21. 33 ± 8. 02） and migration distance（ 303. 27 ± 47. 58） μm were reduced（ P〈0. 05）.The differences were statistically significant（ P〈0. 05）. Conclusion Lentivirus- mediated shRNA targeting DNMT1 can effectively knock down the expression of DNMT1 and up- regulate the expression of T- cadherin,inhibit the proliferation of SW620 cells,promote their apoptosis,reduce their invasion and migration ability.

关 键 词：DNA甲基转移酶1 慢病毒 结肠癌 生物学行为 

分 类 号：R735.35[医药卫生—肿瘤]